A B S T R A C TFatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2 g/L fatty alcohols in shake flasks, and 6.0 g/L in fed-batch fermentation, corresponding to~20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7 g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acidderived products.

Background Lignocellulosic material is a suitable renewable carbon and energy source for microbial cell factories, such as Yarrowia lipolytica. To be accessible for microorganisms, the constituent sugars need to be released in a hydrolysis step, which as a side effect leads to the formation of various inhibitory compounds. However, the effects of these inhibitory compounds on the growth of Y. lipolytica have not been thoroughly investigated. Results Here we show the individual and combined effect of six inhibitors from three major inhibitor groups on the growth of Y. lipolytica. We engineered a xylose consuming strain by overexpressing the three native genes XR, XDH, and XK and found that the inhibitor tolerance of Y. lipolytica is similar in glucose and in xylose. Aromatic compounds could be tolerated at high concentrations, while furfural linearly increased the lag phase of the cultivation, and hydroxymethylfurfural only inhibited growth partially. The furfural induced increase in lag phase can be overcome by an increased volume of inoculum. Formic acid only affected growth at concentrations above 25 mM. In a synthetic hydrolysate, formic acid, furfural, and coniferyl aldehyde were identified as the major growth inhibitors. Conclusion We showed the individual and combined effect of inhibitors found in hydrolysate on the growth of Y. lipolytica. Our study improves understanding of the growth limiting inhibitors found in hydrolysate and enables a more targeted engineering approach to increase the inhibitor tolerance of Y. lipolytica. This will help to improve the usage of Y. lipolytica as a sustainable microbial cell factory.

There is a need for development of sustainable production processes for production of fats/ oils and lipid derived chemicals. The dimorphic oleaginous yeast Yarrowia lipolytica is a promising organism for conversion of biomass hydrolysate to lipids, but in many such processes hyphae formation will be problematic. We have therefore constructed and compared the performance of strains carrying deletions in several published gene targets suggested to abolish hyphae formation (MHY1, HOY1 and CLA4). The MHY1-deletion was the only of the tested strains which did not exhibit hyphae formation under any of the conditions tested. The MHY1-deletion also had a weak positive effect on lipid accumulation without affecting the total fatty acid composition, irrespective of the nitrogen source used. MHY1 has been suggested to constitute a functional homolog of the stress responsive transcription factors MSN2/4 in Saccharomyces cerevisiae, the deletion of which are highly stress sensitive. However, the deletion of MHY1 displayed only minor difference on survival of a range of acute or long term stress and starvation conditions. We conclude that the deletion of MHY1 in Y.lipolytica is a reliable way of abolishing hyphae formation with few detectable negative side effects regarding growth, stress tolerance and lipid accumulation and composition.

The development of a cost-competitive bioprocess requires that the cell factory converts the feedstock into the product of interest at high rates and yields. However, microbial cell factories are exposed to a variety of different stresses during the fermentation process. These stresses can be derived from feedstocks, metabolism, or industrial production processes, limiting production capacity and diminishing competitiveness. Improving stress tolerance and robustness allows for more efficient production and ultimately makes a process more economically viable. This review summarises general trends and updates the most recent developments in technologies to improve the stress tolerance of microorganisms. We first look at evolutionary, systems biology and computational methods as examples of non-rational approaches. Then we review the (semi-)rational approaches of membrane and transcription factor engineering for improving tolerance phenotypes. We further discuss challenges and perspectives associated with these different approaches.